The disappointing results of the recent Merck vaccine clinical phase IIb trials, which utilized adenovirus-based HIV-1 candidates capable of generating an anti-HIV CTL response (HIV vaccine failure prompts Merck to halt trial. Nature 449, 390 (2007), http://www.hvtn.org/science/1107.html) highlights the importance of understanding the mechanisms through which HIV successfully evades the anti-HIV CTL response. In addition, the inability of current therapies to cure HIV disease underlines the importance of taking new approaches at targeting HIV. Existing drugs effectively prevent new infections, however none of the currently available anti- HIV therapies aid in the eradication of pre-existing infected cells. Thus, it is more clear than ever before, that we need to fully understand the viral mechanisms for immune escape to eradicate infected cells, cure disease and prevent new infection. To this end, the HIV Nef protein is an important and under-developed drug target. HIV-1 Nef has been shown to protect infected primary T cells from CTL recognition and killing in vitro by downmodulating MHC-I protein [7]. There is also substantial in vivo evidence that MHC-I downmodulation is critically important in SIV-infected monkeys for HIV disease pathogenesis [8, 9]. Over the past two funding periods of this grant, we have generated substantial evidence indicating that Nef-mediated MHC-I downmodulation occurs in the following manner: Nef binds to immature forms of MHC-I by targeting hypophosphorylated MHC-I cytoplasmic tail domains in the ER/early Golgi. Binding prevents phosphorylation of the MHC-I cytoplasmic tail and disrupts MHC-I transport from the trans-Golgi network (TGN) to the cell surface. MHC-I is then targeted for degradation in lysosomes. Targeting of MHC-I into the endo-lysosomal pathway from the TGN requires the activity of two cellular co-factors, AP-1 and 2-COP. These cellular trafficking proteins bind the Nef-MHC-I complex at distinct sub-cellular locations and promote targeting into the endosomal and lysosomal pathways respectively. Moreover, we now have a detailed understanding of which amino acids are necessary to form the Nef-MHC-I-AP-1 three-way complex. In the up-coming funding period, we will continue to uncover the mechanism by which Nef functions by determining how Nef is able to bypass the normal cellular regulation of these molecules. In addition, we will begin to apply our assay systems to other Nef targets to compare and contrast the manner in which Nef affects other cellular pathways. Finally, we will demonstrate to what extent these Nef activities are generalized across HIV subtypes.